Acta Protozool. (2003) 42: 165 - 169
Short Communication
Extracellular Calcium Changes the Morphology of Induced Pinocytosis
in Amoeba proteus
Wanda KŁOPOCKA, Anna WASIK and Lucyna GRĘBECKA
Department of Cell Biology, Nencki Institute of Experimental Biology, Warszawa, Poland
Summary. The morphology of pinocytosing Amoeba proteus induces by two monovalent cations: Na+ and K+ were examined at different
calcium concentration. It was demonstrated that pinocytotic response of amoeba (number, size and shape of pinocytotic pseudopodia) was
related to the amount of Ca2+ accumulated on the cell surface.
Key words: amoebae, [Ca2+]e, endocytosis.
INTRODUCTION
In Amoeba proteus a typical induced pinocytosis is
manifested after external application of different agents:
aminoacides, proteins, monovalent cations. This pinocytosis is a kind of fluid-phase endocytosis driven by
cortical cytoskeleton and may be compared to
macropinocytosis in Dictyostelium discoideum in which
large fluid-filled vesicles are formed (Maniak 2002).
During induced pinocytosis A. proteus attains a rosette
form with specialized pinocytotic pseudopodia containing channel-like invaginations. The morphology of
pinocytosing amoebae: number, shape, and size of pinocytotic pseudopodia and polarity of their distribution
on the cell surface vary depending on the kind of
Address for correspondence: Wanda Kłopocka, Department of
Cell Biology, Nencki Institute of Experimental Biology, ul. Pasteura
3, 02-093 Warszawa, Poland; E-mail: [email protected]
inducer, as it was demonstrated for two monovalent
cations: Na+ and K+ (Grębecka and Kłopocka 1986).
Since Na+ and K+ can also exert variable effects on the
pattern of Ca2+ shifting between the surface of amoeba
and the medium during induction of pinocytosis (Kłopocka
et al. 2000), it seems possible that there is a relationship
between the type of pinocytotic reaction and the behaviour
of calcium at the cell surface.
MATERIALS AND METHODS
Amoeba proteus was cultured in Pringsheim medium with standard concentration of Ca2+ (0.85 mM) and fed twice a week on
Tetrahymena pyriformis. Before experiments cells were starved for
2 days. All experiments were carried out at room temperature.
The course of pinocytosis was compared in amoebae exposed to
different concentrations of usually used pinocytotic inducers: KCl
and NaCl, in the standard Pringsheim medium, and in Pringsheim
solution with modified [Ca2+]. Pinocytosis was induced by 30 mM
KCl, 125 mM KCl and 125 mM NaCl in standard concentration of
166 W. Kłopocka et al.
Ca2+ (0.85 mM), by 125 mM KCl in [Ca2+]e increased up to 4.85 mM,
and by 30 mM KCl and 125 mM NaCl in the presence of 10 mM
EGTA. Calcium concentration either was changed before application
of the inducer or during pinocytosis.
Changes in the amount of cell-associated Ca2+ during pinocytotic
induction were assessed by adding 45Ca2+ (0.1 mCi/ml, Amersham
Life Science, Little Chalfont, Buckinghamshire, England) according to
the procedure described earlier (Kłopocka et al. 2000). Calcium shifts
between the cell surface and the surrounding medium are shown as
per cent increase or decrease of cell-associated radioactivity during
the experiment. Pinocytosis was controlled 7, 12 and 17 min after
application of the inducer.
Observations were carried out in the differential interference
contrast (Pluta system, PZO Warsaw) and recorded on the tape with
a Panasonic wv BL 600 camera and NV-8051 Panasonic time-lapse
video recorder. Selected frames of various pinocytotic forms were
stored in an IBM PC compatible computer memory. The morphology of pinocytosis is shown by scanning electron microscopy (SEM)
of amoebae fixed and processed as it was previously described
elsewhere (Grębecki et al. 2001).
RESULTS AND DISCUSSION
It was the purpose of present investigations to reveal
whether there is a relationship between the decrease or
increase of calcium amount on the cell surface and the
pinocytotic response induced by two different monovalent cations: K+ and Na+.
Pinocytotic reaction of amoeba was graduated and
characterised by a distinct polarity (the first pinocytotic
structures appeared in the uroidal region, next ones at
the former fronts) and by the development of numerous
small pseudopodia with thin channels, after application of
125 mM NaCl (Fig. 1) or 30 mM KCl (Fig. 2) under
standard medium conditions, and during pinocytosis induced by 125 mM KCl in the medium with [Ca2+]e
increased up to 4.85 mM (not shown). It means that this
kind of pinocytosis occurred, when Ca2+ was binding to
amoebae during stimulation, more or less similar as to the
control cells (Fig. 5).
Stimulation by 125 mM K+ applied in standard
Pringsheim medium caused immediate cell contraction,
the first pinocytotic pseudopodia were formed in any
place of strongly deformed cells and, as a result, amoebae produced few large pseudopodia with very wide
channels (Fig. 3). This was accompanied with a decrease of [Ca2+]e associated to the surface of amoeba
(Fig. 5). Similar coincidence between the alteration of
the morphological features of pinocytosis and the displacement of calcium from the cell surface could be
produced as well during pinocytosis induced by 125 mM
NaCl and 30 mM KCl by Ca2+e chelating by EGTA (not
shown).
On the contrary, addition of 4 mM CaCl2 during
pinocytosis induced by 125 mM K+, that is increasing
[Ca2+]e from 0.85 mM up to 4.85 mM, caused immediate
association of more calcium with the cell surface (Fig. 5)
accompanied by changes in the morphology of pinocytosis. Within 30s after application of calcium, the
pinocytosing cells with few and large pseudopodia (such
as shown in Fig. 3) became rosettes with numerous,
small pseudopodia with thin channels (Fig. 4).
In general, it may be concluded that changes in the
concentration of extracellular calcium associated during
pinocytosis with the cell membrane outer surface and/or
with glycocalyx caused immediate alterations of size and
number of existing pinocytotic structures.
One can suggest that the course of events during
macropinocytosis in Amoeba proteus depends on the
direction of extracellular calcium shifting between amoebae and the medium during cells stimulation. It was
postulated that extracellular calcium plays an important
role in controlling the physiological state of the plasma
membrane in amoebae (Brandt and Freeman 1967,
Brandt and Hendil 1972, Kukulis et al. 1986). Low
concentration of Ca2+e in the presence of other cations
can induce such cellular activities as endocytotic membrane internalisation (Marshall and Nachmias 1965, Hendil
1971, Braatz-Schade and Haberey 1975, Josefsson 1975,
Stockem 1977), whereas high concentration of Ca2+e has
a stabilising effect on the membrane (Gingell 1972). In
our experiments neither 125 mM Na+ nor 30 mM K+
could not induce pinocytosis in A. proteus in [Ca2+]e
increased up to 4.85 mM.
The polarity of pinocytosis is probably related to
stability of the cell membrane. When externally bound
Ca2+ is substituted by the inducer, as it takes place in
125 mM K+ in standard medium, the membrane potential
immediately decreases around the whole cell (BraatzSchade and Haberey 1975, Josefsson et al. 1975) and
the first pinocytotic pseudopodia can develop in any
place of the surface of amoeba. If calcium is not
displaced from the cell surface by the inducing agent, it
is in 125 mM Na+ or 30 mM K+ in the standard medium
and 125 mM K+ in the increased [Ca2+]e, the first
channels are formed at the uroid where suitable conditions for a permanent pinocytosis exist (WohlfarthBottermann and Stockem 1966), because of the strong
membrane folding (Czarska and Grębecki 1966) and its
low stability in this area (Batueva 1965a, b; Bingley
Calcium effect on A. proteus pinocytosis 167
Figs 1-4. Morphology of Amoeba proteus pinocytosis shown in SEM. In all pictures arrowheads indicate pinocytotic structures. Scale bar on
Fig. 1 applies to all figures. 1- pinocytosis 7 min after induction by 125 mM NaCl introduced to the standard Pringsheim medium.
2 - pinocytosis induced for 7 min by low (30 mM) concentration of KCl in the culture medium. 3 - pinocytosis stimulated 7 min by high
(125 mM) concentration of KCl in the culture medium. 4 - pinocytosing amoeba initially stimulated 7 min by 125 mM KCl, and then followed
by addition of 4 mM CaCl2. Insets in Figs 1, 2, 4 are digitally enlarged 3.5 x.
1966). Next channels are formed at the front when it
begins to contract (Grębecka and Kłopocka 1985,
Grębecka 1988) and similar conditions as in uroid are
established.
Calcium also controls cortical cytoskeleton functions
that are responsible for cell shape changes and pseudopodia formation: reorganisation of cortical cytoskeleton in
amoebae (Hellewel and Taylor 1979, Taylor and
Fechheimer 1982, Bray 1992) and interactions of actin
microfilaments with the plasma membrane (Taylor et al.
1980, Kawakatsu et al. 2000), and thus it may influence
the course of pinocytosis. According to the view of
Maeda and Kawamoto (1986) a low [Ca2+] activates
pinocytosis in Dictyostelium discoideum because under
such conditions the content of actin in the cell cortex and
consequently the number of membrane-associated microfilaments are reduced, which may restrict the plasma
membrane flexibility, necessary for pinocytotic structures formation. Our results seem to confirm this
hypothesis and indicate that membrane flexibility in
A. proteus may influence the number and size of
pinocytotic structures.
According to Klein et al. (1988) the size of pinocytotic pseudopodia and the diameter of channels are
168 W. Kłopocka et al.
Fig. 5. Per cent changes in the increase or decrease of [Ca2+]e associated with control cells, during pinocytosis induced by 125 mM NaCl,
30 mM KCl and 125 mM KCl under standard conditions in Pringsheim medium, and 125 mM KCl followed by application of 4 mM CaCl2
(asterisk indicates the moment of calcium application).
regulated by the mode of membrane-microfilament interaction. Pinocytotic pseudopodia are developed due to
the circular detachment of the microfilament layer from
the plasma membrane with the exception of a central
region of fast contact (Stockem et al. 1983, Grębecki
1991). The diameter of the channels depends on the
extent of the areas of contact between the microfilament
layer and the internal face of the plasma membrane
(Klein et al. 1988). The size of pseudopodia is related to
the area of detachment and probably the contraction
degree. It seems possible that Ca2+e can control, via the
membrane of amoeba, the rearrangement of actin cortical network and its attachment to the cell surface.
Conclusions:
1. The pinocytotic response of Amoeba proteus is
related to the amount of Ca2+ accumulated on the cell
surface during the induction of this phenomenon.
2. The direction of calcium shifting during the initiation of pinocytosis depends on the strength of inducer, its
concentration, and the concentration of extracellular
calcium.
Acknowledgement. Authors wish to express their sincere gratitude
to Prof. A. Grębecki for his constructive suggestions for improvement and modification of the manuscript. Authors also thank to Dr.
P. Pomorski for his help in preparation of figures.
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Received on 28th March, 2003; accepted on 31st March, 2003